Method for eliciting an immune response to an immunogen

ABSTRACT

The invention relates to methods for eliciting an immune response to an immunogen, and in particular, to such methods using polymersomes as carriers for immunogen.

CROSS-REFERENCE TO RELATED APPLICATION

This patent application is a continuation of U.S. patent applicationSer. No. 14/646,008, filed May 19, 2015, which is a U.S. National PhaseApplication under 35 U.S.C. § 371 of International Application No.PCT/SG2013/000478, filed Nov. 11, 2013, entitled METHOD FOR ELICITING ANIMMUNE RESPONSE TO AN IMMUNOGEN, which claims the benefit of priority ofSingapore Patent Application No. 201208483-6, filed Nov. 19, 2012, thecontents of which were incorporated by reference in their entirety forall purposes.

TECHNICAL FIELD

The invention relates to methods for eliciting an immune response to animmunogen, and in particular, to such methods using polymersomes ascarriers for the immunogen.

BACKGROUND

Although immunization is a well-established process, there aredifferences in the response level elicited between different immunogensantigens (used interchangably herein). Of interest, membrane proteinsform a class of antigens that produce a low response level, which inturn means that a large number of membrane proteins are required inorder to generate or elicit an immune response to the desired level.Membrane proteins are notoriously difficult to synthesize and areinsoluble in water without the presence of a detergent. This makes itexpensive and difficult to obtain membrane proteins in sufficientquantity for the purpose of immunization.

Furthermore, membrane proteins require proper folding in order tofunction correctly. The immunogenicity of correctly folded membraneproteins are much better than solubilized membrane proteins, which arenot folded in a physiologically relevant manner. Thus, even thoughadjuvants may be used to boost the immunogenicity of such solubilizedmembrane proteins, it is an inefficient method that does not provide toomuch of an advantage.

In addition, the common procedure to raise antibodies against membraneproteins often require a prior knowledge of their native structurewithin membranes in order to design suitable epitopes that can be usedfor the immunization. This immunization is usually performedindependently using isolated peptides which could adopt conformationsvery differently from the one occurring in the full protein in itsnative membrane habitat. Hence, there is a high risk that the antibodiesraised by the isolated peptides may not recognize the target protein invivo after all.

Although transfected cells and lipid-based systems have been used topresent membrane protein antigens to increase the chances of isolatingantibodies that may efficient in vivo, these systems are often unstable,tedious and costly. Moreover, the current state of the art for suchmembrane protein antigens is to use inactive virus-like particles forimmunization.

Therefore, there remains a need to provide for alternative methods thatovercome, or at least alleviate, the above problems.

SUMMARY

The invention described herein provides a method to present membraneproteins to evoke immune response without addition of known adjuvantsusing proteopolymersomes. The present inventors have surprisingly foundthat by providing the circumferential membrane of a polymersome to allowmembrane proton antigens to properly fold, a stronger immune responsethan free membrane proteins antigens is evoked. Consequently, anincrease in the efficiency of antibody production in a subject, such asa mammalian animal, is achieved. The increase in the efficiency can beattained with or without the use of adjuvants. Because full-length andproperly folded membrane protein antigens are presented, the antibodiesproduced by using the invention described herein would also have ahigher affinity for in vivo membrane protein targets and may able toneutralize the virus if the antibodies are raised virus antigens.

Thus, in accordance with one aspect of the invention, it is disclosed amethod for eliciting in a subject an immune response to an immunogen.The method may include injecting the subject with a compositionincluding a polymersome carrier having a circumferential membrane of anamphiphilic polymer. The composition further includes an immunogenintegrated into the circumferential membrane of the amphiphilic polymerof the polymersome carrier. The immunogen may be a membrane-associatedprotein or lipid antigen.

In another aspect of the invention, there is provided a composition forintradermal, intraperitoneal, subcutaneous, intravenous, orintramuscular injection, or non-invasive administration of an immunogen.The composition may include a polymersome carrier having acircumferential membrane of an amphiphilic polymer. The composition mayfurther include an immunogen integrated into the circumferentialmembrane of the amphiphilic polymer of the polymersome carrier. Theimmunogen may be a membrane-associated protein or lipid antigen. Thecomposition may be used in antibody discovery, vaccine discovery, ortargeted delivery.

BRIEF DESCRIPTION OF THE DRAWINGS

In the drawings, like reference characters generally refer to the sameparts throughout the different views. The drawings are not necessarilydrawn to scale, emphasis instead generally being placed uponillustrating the principles of various embodiments. In the followingdescription, various embodiments of the invention are described withreference to the following drawings.

FIG. 1 shows a standard curve of alpha-hemolysin concentration.

FIG. 2A shows optical density measurements of antibody titers O.D.,measurements using alpha-hemolysin-coated plate. 3 mice were injectedwith alpha-hemolysin-presenting polymersomes (closed squares labeled“Ves-Hemo”) while 3 other mice received empty polymersomes (opentriangles labeled “Ves. alone”) Sera were diluted (1:1000) andanti-mouse HRP-coupled antibody was used as a secondary antibody. Anincreasing anti-alpha-hemolysin titer was observed over time withrepeated immunizations.

FIG. 2B shows the same experiment procedures as FIG. 2A using non-coatedplates. No antibody binding is detected.

FIG. 3 shows optical density measurements of immune response.Alpha-hemolysin-presenting polymersomes (closed squares labeled“Ves+Hemo”) gave a higher response than free alpha-hemolysin (opensquares labeled “Hemo”) up to the 3^(rd) bleed, after which a saturationof the immune response was achieved for both. Interestingly,alpha-hemolysin with adjuvant (open circles labeled “Hemo+adj”) gave alower immune response than free-alpha-hemolysin. Empty polymersomes(open triangles labeled “Ves”) remained non-immunogenic.

FIG. 4 shows optical density measurements of immune response.Alpha-hemolysin-presenting polymersomes (closed squares labeled“Ves-Hemo”) elicited a higher immune response than free alpha-hemolysin(open squares labeled “Free Hemo”) in both the 2^(nd) and 3^(rd) bleedswhen intradermal injection is performed.

FIG. 5 shows a standard curve of hemagglutinin (HA) concentration.

FIG. 6 shows ELISA for HA-inserted and empty (control) polymersomes.

FIG. 7 shows optical density measurements of antibody titers. HApolymersomes (triangles) elicited a higher immune response than free HA(circles) after boost (2^(nd) bleed) at equivalent dose of 100 ng HA(***p<0.001 with respect to PBS group). No antibody binding is detectedfor uncoated wells (data not shown).

DESCRIPTION

The following detailed description refers to the accompanying drawingsthat show, by way of illustration, specific details and embodiments inwhich the invention may be practised. These embodiments are described insufficient detail to enable those skilled in the art to practise theinvention. Other embodiments may be utilized and structural, logical,and electrical changes may be made without departing from the scope ofthe invention. The various embodiments are not necessarily mutuallyexclusive, as some embodiments can be combined with one or more otherembodiments to form new embodiments.

In the present context, polymersomes are vesicles with a polymericmembrane, which are typically, but not necessarily always, formed fromthe self-assembly of dilute solutions of amphiphilic block copolymers,which can be of different types such as diblock and triblock (A-B-A orA-B-C). Polymersomes may also be formed of tetrablock pentablockcopolymers. For triblock copolymers, the central block is often shieldedfrom the environment by its flanking blocks, while diblock copolymersself-assemble into bilayers, placing; two hydrophobic blockstail-to-tail, much to the same effect. In most cases, the vesicularmembrane has an insoluble middle layer and soluble outer layers. Thedriving force for polymersome formation by self-assembly is consideredto be the microphase separation of the insoluble blocks, which tend toassociate in order to shield themselves from contact with water.Polymersomes possess remarkable properties due to the large molecularweight of the constitutent copolymers. Vesicle formation is favored uponan increase in total molecular weight of the block copolymers. As aconsequence, diffusion of the (polymeric) amphiphiles in these vesiclesis very low compared to vesicles formed by lipids and surfactants. Owingto this less mobility of polymer chains aggregated in vesicle structure,it is possible to obtain stable polymersome morphologies. Unlessexpressly stated otherwise, the term “polymersome” and “vesicle”, asused hereinafter, are taken to be analogous and may be usedinterchangably.

In the present context, an antigen is any substance that may bespecifically bound by components of the immune system and only antigensthat are capable of eliciting (or evoking or inducing) an immuneresponse are said to be immunogenic and are called immunogens. Membraneproteins form a class of antigens that produce a low response level. Ofspecific interest, membrane-associated proteins (i.e. the antigensmentioned herein) are integrated (or incorporated or carried) into thewall of a polymersome, allowing the membrane-associated proteins to befolded in a physiologically relevant manner (i.e. presently termed aspolymersome carrier or antigen-presenting polymersome, both terms usedinterchangably hereinafter). This greatly boosts the immunogenicity ofthe membrane proteins so that when compared to free membrane proteins, asmaller amount of membrane proteins can be used to produce the samelevel of immune response. Furthermore, the larger size of thepolymersomes (compared to free membrane proteins) allows them to bedetected by the immune system more easily.

Since the immunization is performed using the full protein (instead offragment thereof) in a synthetic environment that allows its properfolding, the probability of isolating antibodies that are capable ofdetecting the membrane protein in vivo would be higher. Moreover, theimmunization and antibody generation can be performed without any priorknowledge of the membrane protein structure, which is otherwisenecessary when using a peptide-based immunization approach.

Further, when compared to other techniques, present approach allowsrapid and cost effective production of membrane protein inserted in astable membrane environment.

Thus, in accordance with one aspect of the present invention, a methodfor eliciting in a subject an immune response to an immunogen isdisclosed. The method may include injecting the subject with acomposition including polymersome carrier having a circumferentialmembrane of an amphiphilic polymer. The composition further includes animmunogen integrated into the circumferential membrane of theamphiphilic polymer of the polymersome carrier. The immunogen is amembrane-associated protein. Alternatively, the immunogen may also be alipid antigen. In such embodiments, the immunogen may be a syntheticlipid or a natural lipid.

The frequency of the injection may be determined and adjusted by aperson skilled in the art, dependent on the level of response desired.For example, weekly or bi-weekly injections of the polymersome carriersmay be given to the subject, which may include a mammalian animal. Theimmune response can be measured by quantifying the blood concentrationlevel of antibodies in the mammalian animal against the initial amountof immunogens carried by the polymersome carrier. The structure of thepolymersomes may include amphiphilic block copolymers self-assembledinto a vesicular format and integrated membrane proteins spanning thewall of the vesicles, whereby the membrane proteins are the antigens tobe presented, and are incorporated by method of reconstitution or invitro synthesis or spontaneous insertion. The membrane proteins can bereconstituted with the aid of detergents, surfactants, temperaturechange or PH change. The vesicular structure provided by the amphiphilicblock copolymers allows the membrane protein antigens to be folded in aphysiologically correct and functional manner, allowing the immunesystem of the target mammalian animal to detect said antigens, therebyproducing a strong immune response.

In various embodiments the injection of the composition may includeintraperitoneal, subcutaneous, or intravenous, intramuscular injection,or non-invasive administration.

In alternative embodiments, the injection of the composition may includeintradermal injection. It has been surprisingly found by the inventorsin experiments involving intradermally injected mice thatalpha-hemolysin presenting polymersomes are much more efficient thanfree alpha-hemolysin in eliciting immune response, where thealpha-hemolysin presenting polymersomes elicited a higher immuneresponse than free alpha-hemolysin in the 2^(nd) and 3^(rd) bleed (seeExamples section below).

The immune response level may be further heightened or boosted byincluding an adjuvant in the composition including the polymersomecarrier carrying the immunogen. In such embodiments, the polymersomeearlier carrying the immunogen and the adjuvant are administeredsimultaneously to the subject.

In alternative embodiments, the adjuvant may be administered separatelyfrom the administration of the composition including the polymersomecarrier carrying the immunogen. The adjuvant may be administered before,simultaneously, or after the administration of the composition includingthe polymersome carrier carrying the immunogen. For example, theadjuvant may be injected to the subject after injecting the compositionincluding the polymersome carrier carrying the immunogen.

A person skilled in the art would readily recognize and appreciate thatthe types of adjuvant to be injected depend on the types of immunogen tobe injected. The immunogen may be an antigen of bacterial, viral orfungi origin. For example, in the case where the antigen isalpha-hemolysin, the adjuvant may be complete Freund adjuvant. Otherantigen-adjuvant pairs are also suitable for use in the present method.In certain embodiments, the use of adjuvants is not needed. In yetcertain embodiments, the present method works better, i.e. strongerimmune response being evoked, without the use of adjuvants.

In other embodiments, the membrane-associated protein may be atransmembrane protein, G protein-coupled receptor, neurotransmitterreceptor kinase, porin, ABC transporter, ion transporter, acetylcholinereceptor and cell adhesion receptor. The membrane proteins may also becoupled with a tag or may be tag-free. If the membrane proteins aretagged, then the tag may be selected from epitopes such as VSV, His tag,Strep tag, Flag tag, Intein tag or GST tag or a partner of a highaffinity binding pair such as biotin avidin or from a label such as afluorescent label, an enzyme label, NMR label or isotope label.

The membrane proteins is may be presented prior to incorporation, orincorporated simultaneously with the production of the protein through acell-free expression system. The cell-free expression system may be anin vitro transcription and translation system.

The cell-free expression system may also be an eukaryotic cell-freeexpression system such as the TNT® system based on rabbit reticulocytes,wheat germ extract or insect extract, a prokaryotic cell-free expressionsystem or an archaic cell-free expression system.

As mentioned above, the polymersomes may be formed of amphiphilicdiblock or triblock copolymers. In various embodiments, the amphiphilicpolymer may include at least one monomer unit of a carboxylic acid, anamide, an amine, an alkylene, a dialkylsiloxane, an ether or an alkylenesulphide.

In certain embodiments, the amphiphilic polymer may be a polyether blockselected from the group consisting of an oligo(oxyethylene) block, apoly(oxyethylene) block, an oligo(oxypropylene) block, apoly(oxypropylene) block, an oligo(oxybutylene) block and apoly(oxybutylene) block. Further examples of blocks that may be includedin the polymer include, but are not limited to, poly(acrylic acid),poly(methyl acrylate), polystyrene, poly(butadiene),poly(2-methyloxazoline), poly(dimethyl siloxane), poly(e-caprolactone),poly(propylene sulphide), poly(N-isoproylacrylamide),poly(2-vinylpyridine) poly(2-(diethylamino)ethyl methacrylate),poly(2-(diisopropylamino)ethylmethacrylate),poly(2-(methacryloyloxy)ethylphosphorylcholine) and poly(lactic acid).Examples of a suitable amphiphilic polymer include, but are not limitedto, poly(ethyl ethylene)-b-poly(ethylene oxide) (PEE-b-PEO),poly(butadiene)-b-poly(ethylene oxide) (PBD-b-PEO),poly(styrene)-b-poly(acrylic acid) (PS-PAA),poly(2-methyloxazoline)-b-poly(di-methylsiloxane)-b-poly(2-methyloxazoline)(PMOXA-b-PDMS-b-PMOXA),poly(2-methyloxa-zoline)-b-poly(dimethylsiloxane)-b-poly(ethylene oxide)(PMOXA-b-PDMS-b-PEO), poly(ethylene oxide)-b-poly(propylenesulfide)-b-poly(ethylene oxide) (PEO-b-PPS-b-PEO) and a poly(ethyleneoxide)-b-poly(buylene oxide) block copolymer. A block copolymer can befurther specified by the average block length of the respective blocksincluded in a copolymer. Thus PB_(M)PEO_(N) indicates the presence ofpolybutadiene blocks (PB) with a length of M and polyethyleneoxide (PEO)blocks with a length of N, M and N are independently selected integers,which may for example be selected in the range from about 6 to about 60.Thus PB₃₅PEO₁₈indicates the presence of polybutadiene blocks with anaverage length of 35 and of polyethyleneoxide blocks with an averagelength of 18. In certain embodiments, the PB-PEO diblock copolymercomprises 5-50 blocks PB and 5-50 blocks PEO. Likewise, PB₁₀PEO₂₄indicates the presence of polybutadiene blocks with an average length of10 and of polyethyleneoxide blocks with an average length of 24. As afurther example E_(O)B_(P) indicates the presence of ethylene blocks (E)with a length of O and butylene blocks (B) with a length of P, O and Pare independently selected integers, e.g. in the range from about 10 toabout 120. Thus E₁₆B₂₂ indicates the presence of ethylene blocks with anaverage length of 16 and of butylene blocks with an average length of22.

In certain embodiments, the polymersome carrier may contain one or morecompartments (or otherwise termed “multicompartments”).Compartmentalization of the vesicular structure of polymersome allowsfor the co-existence of complex reaction pathways in living cell andhelps to provide a spatial and temporal separation of many activitiesinside a cell. Accordingly, more than one type of immunogen may beincorporated in the polymersome carrier. The different immunogens mayhave the same or different isoforms. Each compartment may also be formedof a same or a different amphiphilic polymer. In various embodiments,two or more different immunogens are integrated into the circumferentialmembrane of the amphiphilic polymer. Each compartment may encapsulate atleast one of peptide, protein, and nucleic acid. The peptide, protein,or nucleic acid may be immunogenic.

In the case where the polymersome carrier contains more than onecompartment, the compartments may comprise an outer block copolymervesicle and at least one inner block copolymer vesicle, wherein the atleast one inner block copolymer vesicle is encapsulated inside the outerblock copolymer vesicle. In some embodiments, each of the blockcopolymer of the outer vesicle and the inner vesicle includes apolyether block such as a poly(oxyethylene) block, a poly(oxypropylene)block, and a poly(oxybutylene) block. Further examples of blocks thatmay be included in the copolymer include, but are not limited to,poly(acrylic acid), poly(methyl acrylate), polystyrene, poly(butadiene),poly(2-methyloxazoline), poly(dimethyl siloxane),poly(L-isocyanoalanine(2-thiophen-3-yl-ethyl)amide),poly(e-caprolactone), poly(propylene sulphide),poly(N-iscpropylacrylamide), poly(2-vinylpyridine),poly(2-(diethylamino)ethyl methacrylate),poly(2-(diisopropylamino)ethylmethacrylate),poly(2-(methacryloyloxy)ethylphosphorylcholine) and poly(lactic acid).Examples of suitable outer vesicles and inner vesicles include, but arenot limited to, poly(ethyl ethylene)-b-poly(ethylene oxide) (PEE-b-PEO),poly(butadiene)-b-poly(ethylene oxide) (PBD-b-PEO),poly(styrene)-b-poly(acrylic acid) (PS-b-PAA), poly(ethyleneoxide)-poly(caprolactone) (PEO-b-PCL), poly(ethylene oxide)-poly(lacticacid) (PEO-b-PLA), poly(isoprene)-poly(ethylene oxide) (PI-b-PEO),poly(2-vinylpyridine)-poly(ethylene oxide) (P2VP-b-PEO), poly(ethyleneoxide)-poly(N-isopropylacrylamide) (PEO-b-PNIPAm), poly(ethyleneglycol)-poly(propylene sulfide) (PEG-b-PPS),poly(methylphenylsilane)-poly(ethylene oxide)(PMPS-b-PEO-b-PMPS-b-PEO-b-PMPS),poly(2-methyloxazoline)-b-poly-(dimethylsilaxane)-b-poly(2-methyloxazoline)(PMOXA-b-PDMS-b-PMOXA),poly(2-methyloxa-zoline)-b-poly(dimethylsiloxane)-b-poly(ethylene oxide)(PMOXA-b-PDMS-b-PEO),poly[styrene-b-poly(L-isocyanoalanine(2-thiophen-3-yl-ethyl)amide](PS-b-PIAT), poly(ethylene oxide)-b-poly(propylenesulfide)-b-poly(ethylene oxide) (PEO-b-PPS-b-PEO) and a poly(ethyleneoxide)-poly(buylene oxide) (PEO-b-PBO) block copolymer. A blockcopolymer can be further specified by the average number of therespective blocks included in a copolymer. Thus PS_(M)-PIAT_(N)indicates the presence of polystyrene blocks (PS) with M repeating unitsand poly(L-isocyanoalanine(2-thiophen-3-yl-ethyl)amide) (PIAT) blockswith N repeating units. M and N are independently selected integers,which may for example be selected in the range from about 5 to about 95.Thus PS₄₀-PIAT₅₀ indicates the presence of PS blocks with an average of40 repeating units and of PIAT blocks with an average of 50 repeatingunits.

By “encapsulated” it is meant that the inner vesicle is completelycontained inside the outer vesicle and in surrounded by the vesicularmembrane of the outer vesicle. The confined space surrounded by thevesicular membrane of the outer vesicle forms one compartment. Theconfined space surrounded by the vesicular membrane of the inner vesicleforms another compartment.

Further details of suitable multicompartmentalized polymersomes can befound in PCT Publication No. WO 2012/018306, the contents of which beinghereby incorporated by reference in its entirety bar all purposes.

The polymersomes may also be free-standing or immobilized on a surface,such as those described in PCT Publication No. WO 2010/123462, thecontents of which being hereby incorporated by reference in its entiretyfor all purposes.

In additional embodiments, a secondary protein that complexes with themembrane protein antigen may be encapsulated or incorporated in thelumen of the polymersome carrier. Advantageously, the secondary proteinstabilises the membrane protein antigen in a specific conformation.

In another aspect of the invention, there is provided a composition forintradermal intraperitoneal, subcutaneous, intravenous, or intramuscularinjection, or non-invasive adminstration of an immunogen. Thecomposition may include a polymersome carrier having a circumferentialmembrane of an amphiphilic polymer. The composition may further includean immunogen integrated into the circumferential membrane of theamphiphilic polymer of the polymersome carrier. The immunogen may be amembrane-associated protein or lipid antigen. The composition may beused in antibody discovery, vaccine discovery, or targeted delivery.

In summary, the present invention demonstrates for the first time theuse of membrane proteins incorporated within polymersomes to generate animmune response. Although a combination of polymersomes and membraneprotein antigens have been used to elicit an immune response previously,the polymersomes were used with known adjuvant molecules, and themembrane proteins were not presented in a physiologically relevantmanner. On the other hand, the present polymersome carriers are not usedas an adjuvant; rather the polymersome carriers are used as vehicles toaccommodate the membrane protein antigens, thereby allowing properfolding of the membrane protein antigens therein, which consequentlyenables a better immune response to be evoked than conventionaldetergent solubilized membrane protein antigens.

Compared to existing techniques, present invention offers the followingadvantages:

-   -   The immune response could possibly be further boosted by using        adjuvants.    -   the polymers are inherently robust, and can be tailored or        functionalized to increase their circulation time in the body.    -   The polymers are cheap and quick to synthesize.    -   The amount of membrane proteins required to elicit an immune        response is lesser.    -   The full length of membrane protein antigen is used, making it        more likely that the antibodies generated will be able to detect        membrane proteins in vivo    -   The membrane proteins can be incorporated into polymersome        carriers via in vitro translation or transcription which is        advantageous to raise antibodies against difficult membrane        protein antigens.

With these advantages, the invention described herein provides a methodto evoke an immune response from membrane protein antigens that isfaster, cheaper, more accurate, and simpler than current state of theart. Possible applications of present invention include production ofantibodies in vaccination and generating therapeutic antibodies.

In order that the invention may be readily understood and put intopractical effect, particular embodiments will now be described by way ofthe following non-limiting examples.

EXAMPLES Example 1

A method for eliciting in mice an immune response to alpha-hemolysinusing alpha-hemolysin-presenting polymersomes is now described in thefollowing paragraphs.

Materials & Methods

Poly(butadiene-b-ethylene oxide) (PBd₂₁-PEO₁₄) BD21 amphiphilic blockcopolymer was purchased from Polymer Source (Canada). Alpha-hemolysinfrom staphylococcus aureus, 3-(N-morpholino) propanesulfonic acid(MOPS), Tris(hydroxymethyl)aminomethane hydrochloride (Tris), magnesiumchloride and sodium chloride were all bought from Sigma Aldrich(Singapore), Tetrahydrofuran (THF) was purchased from Tedia (Ohio, USA).

Preparation of Polymersomes

The polymersomes were prepared by film rehydration method. BD21 polymerwas dissolved in THF, and dried as a thin film on the wall of a conicalbottom glass tube under a stream of nitrogen gas. The polymer film isfurther dried under vacuum. Subsequently, ultrapure water was added tothe tube and stilled to rehydrate the film and allow spontaneousformation of polymer vesicles, resulting in a uniformly turbid solution.The resulting vesicle dispersion was then extruded with 0.45 μm PVDFfilters (Millipore), and dialysis against ultrapure water was carriedout to remove any remaining solvent. Alpha-hemolysin was dissolved inMOPS-NaCl buffer (0.01M MOPS, 0.1M NaCl, pH 7).

The polymersomes were formed by adding an aliquot of alpha-hemolysinsolution to polymer vesicle dispersion. The mixture was then incubatedto allow reconstitution of alpha-hemolysin into the polymersomes.Subsequently, free alpha-hemolysin was separated fromalpha-hemolysin-presenting polymersomes using centrifugal filters. Thealpha-hemolysin-presenting polymersomes were then resuspended in 50 ulof TMN buffer (100 mM Tris, 50 mM MgCl₂ and 100 mM NaCl, adjusted to pH7.5).

Quantification of Alpha-Hemolysin Concentration

The quantification of the amount of alpha-hemolysin inserted intoalpha-hemolysin-presenting polymerosomes was determined using a standardcurve obtained from known concentrations of free hemolysin (FIG. 1). 100ng/well of polyclonal anti-alpha-hemolysin antibodies in coating buffer(0.1M CO₃/HCO₃pH9-9.8) were coated in a 96-well plate overnight at 4° C.The day after, the wells were blocked in blocking buffer (1% BSA in 1XPBS) at room temperature (RT) for 1 h. Different concentrations freealpha-hemolysin starting from 500 ng/100 ul in blocking buffer were thenincubated for 1 h, RT. Additionally, alpha-hemolysin-presentingpolymersome samples were prepared in the same buffer (1:10 dilution) andincubated similarly. Subsequently, anti-alpha-hemolysin mouse serum(1:1000) in blocking buffer was applied, followed by anti-mouseHRP-coupled antibodies (1:4000). Peroxidase activity was quantifiedthrough triplicate measurements with TMB substrate. Triplicatemeasurements were also performed on non-alpha-hemolysin-coated wells todetermine non-specific binding (NSB). Extrapolation using the obtainedstandard curve allowed us to estimate the amount of insertedalpha-hemolysin proteopolymerosomes to be 1.32 μg/ml+/−0.6 (n=4).

Injection of Polymersomes

100-150 ng per mouse of alpha-hemolysin-presenting proteopolymersomeswere injected intraperitoneally or intradermally in C57B/6 mice (3 miceper group) as follows: 1^(st) boost on day 1, 2^(nd) boost on day 14followed by a boost every 7 days for 4 more weeks. Blood samples werecollected before each immunization using capillaries sampling from themice's cheek.

Quantification of Immune Response

100 ng per well of free alpha-hemolysin in boating buffer were coated ina 96-well plate overnight. The wells were blocked using blocking bufferthe day after. Each blood sample was diluted (1:100 or 1:1000) inblocking buffer and incubated on alpha-hemolysin-coated or non-coatedwells for 1 h at RT. After 3 washes (PBS 1X), anti-mouse HRP-coupledantibodies (1:4000) were incubated for 1 h at RT followed by 3 washesand TMB substrate reaction.

Results & Discussion

To demonstrate the present method of using, membrane proteinantigen-presenting polymersomes to elicit an immune response,alpha-hemolysin (antigen) was incorporated into BD22 polymersomes toform alpha-hemolysin presenting polymersomes.

To demonstrate the immunogenicity of the alpha-hemolysin-presentingpolymersomes, they were injected intraperitoneally into 3 mice while 3other mice received polymersomes without proteins. The sera weretittered against an alpha-hemolysin-coated plate (FIG. 2A). A robustantibody titer was obtained after the 3^(rd) injection ofalpha-hemolysin-presenting polymersomes while empty polymersomesremained without effect throughout the experiment. No titer was detectedwhen the sera were tested on plates without alpha-hemolysin coating,showing that the antibody titer was specific for alpha-hemolysin (FIG.2B). This shows that alpha-hemolysin presenting polymersomes are able toelicit an immune response, raising antibodies against alpha-hemolysinfor immunization or antibody production, even though the amount ofalpha-hemolysin used (100-150 ng) is much lower than the usual microgramdosage.

To further analyse the immunogenicity of alpha-hemolysin presentingpolymersomes, the polymersomes were injected into 3 mice, while 9 othermice were injected with various controls (3 mice with TMN buffer, 3 micewith free alpha-hemolysin and 3 mice with alpha-hemolysin and completeFreund adjuvant), FIG. 3 shows the immunogenic response of the micethrough 5 injections. As with the previous experiment, alpha-hemolysinpresenting polymersomes were able to elicit an immune response.Surprisingly, free alpha-hemolysin of a comparable amount (100 ng) wasalso able to elicit an immune response without the use of adjuvants,although the response was somewhat lower for the first 3 injections.Free alpha-hemolsyin injected together with an adjuvant led to a lessefficient response while empty polymersomes remained non-immunogenic.Hence, part of the observed immune response obtained usingalpha-hemolysin presenting polymerosomes could be due to immunogenicityof alpha-hemolysin itself. Such immunogenicity without adjuvant was alsoobserved in a study where a truncated version of alpha-hemolysin wasused to immunize mice, although a much higher dosage was used (5 μg).

To analyse the immunogenicity of alpha-hemolysin presenting polymersomeseven further, intradermal injections were carried out. Much attentionhas been focused on immunization performed in different layers of theskin recently, where subcutaneous and intradermal injections areregaining popularity over intraperitoneal and intramuscular routesbecause the skin is enriched in antigen-presenting cells such asdendritic cells and macrophages. In addition, a lot of effort has beenput into developing easy-to-use needle-free devices that will allowvaccine delivery through the skin. Because of this interest, intradermalinjections were carried out in this further experiment. Alpha-hemolysinpresenting polymersomes were injected into 3 mice, while freealpha-hemolysin was injected into 2 mice. FIG. 4 shows the immunogenicresponse of the mice through 3 injections. Interestingly, thealpha-hemolysin presenting polymersomes are now much more efficient thanfree alpha-hemolysin in eliciting an immune response, where thealpha-hemolysin presenting polymersomes elicited a higher immuneresponse than free alpha-hemolysin in the 2^(nd) and 3^(rd) bleed. Itappears that the immunogenicity of free alpha-hemolysin attenuated byintradermal injection while alpha-hemolysin presenting polymerosomes arestill able to generate an immune response. This is probably because therelatively large polymersomes are able to recruit dendritic cellspresent in the dermis.

In conclusion, the inventors have demonstrated a method of usingmembrane protein antigen-presenting polymersomes to elicit an immuneresponse by using alpha-hemolysin presenting polymersomes as an example.The alpha-hemolysin presenting polymersomes were able to elicit animmune response more efficiently than both free alpha-hemolysin andalpha-hemolysin injected with an adjuvant. The membrane protein antigenpresenting polymersomes can be used for both immunization and antibodyproduction.

Example 2

A method for eliciting in mice an immune response usinghemagglutinin-presenting polymersomes is now described in the followingparagraphs.

Preparation of Hemagglutinin Polymersomes

The polymersomes were prepared by film rehydration method, BD21 polymerwas dissolved in chloroform, and dried as a thin film. Subsequently,ultrapure water was added to rehydrate the film and allow spontaneousformation of polymer vesicles. The resulting vesicle dispersion was thenextruded with 0.45 μm and 0.22 μm membranes and dialysed againstultrapure water to remove any remaining solvent.

Recombinant Hemagglutinin (HA) Protein (Influenza A Virus H3N2 Wisconsin67/05, MyBioSource) was supplied as a sterile filtered solution in 10 mMsodium phosphate, pH 7,4,150 M NaCl and 0.005% Tween-20.

The HA polymersomes were formed by adding an aliquot of HA solution topolymer vesicle dispersion. The mixture was then incubated to allowreconstitution HA into the polymersomes. Subsequently, free HA wasseparated from HA-presenting polymersomes using centrifuge filters. TheHA-presenting polymersomes were then resuspended in PBS buffer.

Quantification of HA Concentration

The quantification of the amount of HA inserted nun HA-presentingpolymersomes was determined using a standard curve obtained from knownconcentrations of free HA (FIG. 5). Serial dilutions of free HA,starting at 5 μg/ml and HA polymersomes (FIG. 6) starting at 1:10dilution in coating buffer (0.1M CO₃/HCO₃pH 9-9.8) were coated in a384-well plate overnight at 4° C. The day after, the wells were washedwith PBS and blocked in blocking buffer (1% BSA in 1X PBS, 50 μl/well)at room temperature (RT) for 1 h. Subsequently, anti-HA antibody inblocking buffer was applied, incubated at room temperature (RT) for 1 hand washed with PBS, followed similarly by anti-rabbit HRP-coupledantibodies (1:2.000). Peroxidase activity was quantified throughtriplicate measurement with OPD substrate. Triplicate measurements werealso performed on non-coated wells to determine background signal.Polymersomes without HA inserted were also included as controls toensure the signal obtained was HA-specific. Extrapolation using theobtained standard curve was performed on the dilutions of HApolymersomes giving rise to signal below saturation, which allowed us toestimate the amount of inserted HA in polymerosomes to be 10.908μg/ml+/−1.43 (n=3).

Immunization of Mice with HA Polymersomes

100 ng per mouse of HA soluble or in polymersomes were injectedsubcutaneously in BALB/c mice (3 mice per group) as follows: 1st dose(prime) on day 1 and 2^(nd) dose (boost) on day 21. As a positivecontrol, a group of mice was immunized with 500 ng of HA and adjuvant(complete Freund's adjuvant for prime and incomplete Freund's adjuvantfor boost, prepared at 1:1 volume ratio according to manufacturer'sinstruction) while mice were immunized with PBS as negative control.Serum samples were collected before immunization, immediately before theboost and 1 week after boost using capillaries sampling from the mice'scheek.

Quantification of Immune Response

15 ng per well of free HA in coating buffer were coated in a 384-wellplate overnight at 4° C. The day after, the wells were washed with PBSand blocked in blocking buffer (1% BSA in 1X PBS) at room temperature(RT) for 1 h. Serum samples of individual mice at each time point wasdiluted (1:100, 1:1000 or 1:10000) in blocking buffer and incubated onHA-coated or non-coated wells for 1 h at RT (FIG. 7). After 3 washes(PBS 1X), HRP-coupled anti-mouse IgG (1:4000) were incubated for 1 h atRT followed by 3 washes. Peroxidase activity was then quantified withOPD substrate. HA polymersomes elicited a higher antibody response thanfree HA after boost (2^(nd) bleed) at equivalent dose of 100 ng HA.

Summary

In this example, it has been demonstrated a method of using membraneprotein-presenting polymersomes to elicit an immune response by using HAas a model antigen. The HA polymersomes were able to elicit an antibodyresponse more efficiently than free HA at a equivalent immunizationdose. The membrane protein antigen-presenting polymersomes can bepotentially used for vaccination to induce protective immune responses,as well as for generating antibodies against membrane proteins that areotherwise difficult to induce.

Furthermore, present inventors have used CXCR4 polymersomes to elicitantibodies against CXCR4 to demonstrate the antibodies can be generatedfor membrane protein antigens that are inserted into the circumferentialmembrane via in-vitro synthesis.

Hemagglutination inhibition and microneutralization assay with influenzavirus (same strain) to test the functionality of the antibodiesgenerated in this work are currently undertaken to prove that they areable to neutralize the virus.

It is also currently being investigated to see if the antibodies raisedagainst CXCR4 polymersomes in this study has much more specific affinityto the CXCR4 antigens produced in cells to demonstrate that this can beused as a tool to generate antibodies against membrane protein antigens.

By “comprising” it is meant including, but not limited to, whateverfollows the word “comprising”. Thus, use of the term “comprising”indicates that the listed elements are required or mandatory, but thatother elements are optional and may or may not be present.

“By consisting of” is meant including, and limited to, whatever followsthe phrase “consisting of”. Thus, the phrase “consisting of” indicatesthat the listed elements are required or mandatory, and that no otherelements may be present.

The inventions illustratively described herein may suitably be practicedin the absence of any element or elements, limitation or limitations,not specifically disclosed herein. Thus, for example, the terms“comprising”, “including”, “containing”, etc. shall he read expansivelyand without limitation. Additionally, the terms and expressions employedherein have been used as terms of description and not of limitation, andthere is no intention in the use of such terms and expressions ofexcluding any equivalents of the features shown and described orportions thereof, but it is recognized that various modifications arepossible within the scope of the invention claimed. Thus, it should beunderstood that although the present invention has been specificallydisclosed by preferred embodiments and optional features, modificationand variation of the inventions embodied therein herein disclosed may beresorted to by those skilled in the art, and that such modifications andvariations are considered to be within the scope of this invention.

By “about” in relation to a given numerical value, such as fortemperature and period of time, it is meant to include numerical valueswithin 10% of the specified value.

The invention has been described broadly and generically herein. Each ofthe narrower species and sub-generic groupings falling within thegeneric disclosure also form part of the invention. This includes thegeneric description of the invention with a proviso or negativelimitation removing any subject matter from the genus, regardless ofwhether or not the excised material is specifically recited herein.

Other embodiments are within the following claims and non-limitingexamples. In addition, where features or aspects of the invention aredescribed in terms of Markush groups, those skilled in the art willrecognize that the invention is also thereby described in terms of anyindividual member or subgroup of members of the Markush group.

What is claimed is:
 1. A method of generating an antibody, comprisingadministering to a subject a composition comprising a polymersomecarrier having a circumferential membrane of an amphiphilic polymer anda non-self immunogen integrated into the circumferential membrane of theamphiphilic polymer of the polymersome carrier, wherein the non-selfimmunogen is a membrane-associated protein or lipid antigen, wherein theamphiphilic polymer is a poly(butadiene)-poly(ethylene oxide) (PB-PEO)diblock copolymer or apoly(2-methyloxazoline)-b-poly(dimethylsiloxane)-b(2-methyloxazoline)(PMOXA-PDMS-PMOXA) triblock copolymer, wherein the membrane-associatedprotein is selected from the group consisting of a transmembraneprotein, G protein-coupled receptor, neurotransmitter receptor, kinase,porin, ABC transporter, ion transporter, acetylcholine receptor, and acell adhesion receptor.
 2. The method of claim 1, further comprisingisolating from the subject one or more antibodies generated in responseto the administration of the composition comprising the polymersome. 3.The method of claim 1, wherein the PB-PEO diblock copolymer comprises5-50 blocks PB and 5-50 blocks PEO.
 4. The method of claim 2, whereinthe subject is a mammalian animal.